With hundreds of gene therapy clinical trials ongoing and about a dozen gene therapy products already on the market, we must provide scientists with the tools needed to characterize this new generation of biotherapeutics. Traditional biologics, such as monoclonal antibodies, are manufactured by expressing one protein in a cell line and purifying it. For gene therapy products, viruses, like the adeno-associated virus (AAV), are typically incorporated into the final product as the gene delivery vehicle. With this addition, AAV attributes need to be added to the product and process monitoring toolbox, including AAV purity, identity, and empty and full capsid analysis. With the right analytical tools for complex gene therapy characterization and monitoring through the development process, scientists can more quickly develop safe, high-quality gene therapy products while reducing time-to-market and reducing costs of manufacturing.
Empty/ Partial/ Full Capsid Analysis
AAV vectors are common gene delivery vehicles. Empty capsids, which do not contain the gene therapy of interest, and partial capsids, those containing only a fragment of the gene of interest, are by-products of the AAV production and can impact product safety and efficacy. The amount of full, partial, and empty capsids, therefore, needs to be characterized and monitored through process development. There are various methods used to characterize full, partial, and empty capsid levels including analytical ultracentrifugation, transmission electron microscopy, charge detection mass spectrometry, and anion exchange chromatography.
It is important to understand the capsid identity because each AAV serotype has unique capsid proteins that transfer the genetic material to specific cells or organs. Depending on the therapeutic target area, the correct AAV serotype is chosen and identity and purity are monitored. All AAV capsids consist of three proteins (VP1, VP2, and VP3) that share high-sequence homology, and ensuring the identity and purity of these proteins are critical to viral infectivity and viral transfer. Given the importance of the serotype and the capsid protein composition, there exists a need for methods to identify and monitor the capsid and the capsid proteins through gene therapy development and manufacturing.
Methods in Action: In a paper by Liu et al, capsid proteins from eight AAV serotypes were characterized using HILIC chromatography coupled to a mass spectrometer.
Monitoring intact capsids allows for mass confirmation of empty versus genome packed capsids. To add to the complexity, genome loaded viral vector particles are even more heterogeneous because the package can be a mix of genomic material. Using charge-detection mass spectrometry (CD-MS) opens the door to analyzing these macromolecules with high mass heterogeneity, providing a means to quality control genome packaging in production of gene therapy products.
Methods in Action: In a paper by Worner et al, Orbitrap-based CD-MS was used to successfully resolve distinct distributions of empty and full AAV8 particles.